Nitroreductase bsntr mutant and application thereof

Abstract

This invention relates to a BsNTR mutant nitroreductase and its applications, belonging to the field of biotechnology. The BsNTR mutant is BsNTR-M, and its amino acid sequence is shown in SEQ ID NO.2. This invention designs and modifies wild-type BsNTR-WT from Bacillus subtilis. The resulting mutant BsNTR-M not only exhibits significantly improved thermostability (Tm value 14°C higher than wild-type BsNTR-WT) but also demonstrates 1.6 times enhanced enzyme catalytic activity. This effectively overcomes the technical bottlenecks of poor thermostability, weak environmental tolerance, and limited activity inherent in wild-type enzymes. Furthermore, it possesses excellent expression and purification performance, allowing for efficient expression and purification in prokaryotic expression systems while retaining high specificity for nitro compounds and good biocompatibility and environmental safety. Large-scale production can be achieved without complex equipment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a BsNTR mutant of nitroreductase and its applications. Background Technology

[0002] Nitroreductases (NTRs) are a class of flavin-dependent oxidoreductases widely found in microorganisms and plant and animal cells. They use NAD(P)H as electron donors to specifically catalyze the reduction reactions of nitro compounds, progressively reducing nitro groups (-NO2) to nitroso groups, hydroxylamine, and ultimately amino groups (-NH2). These enzymes are characterized by high catalytic activity, strong substrate selectivity, and mild reaction conditions, making them valuable for applications in the degradation of nitro pollutants, detection of tumor hypoxia markers, activation of fluorescent probes, prodrug activation, and biocatalytic synthesis.

[0003] Nitroreductase (BsNTR) derived from Bacillus subtilis, a natural reductase from Gram-positive bacteria, possesses good biosafety, environmental compatibility, and industrial application potential. Its expression and purification are relatively easy, with few side reactions and a moderate substrate spectrum, showing promising application prospects in biocatalysis, pollutant remediation, and in vivo detection systems. However, wild-type BsNTR exhibits significant drawbacks and technical bottlenecks in practical applications, primarily manifested in poor thermal and structural stability, weak environmental tolerance, and a sharp decline in activity under acidic, alkaline, or high-salt conditions, making it difficult to meet the demands of efficient catalysis, industrial applications, and large-scale production. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a BsNTR mutant of nitroreductase and its application.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides a BsNTR mutant of nitroreductase and its application, wherein the BsNTR mutant is BsNTR-M; the amino acid sequence of BsNTR-M is shown in SEQ ID NO.2.

[0006] As a further optimization of the above invention, the nitroreductase BsNTR mutant is obtained by modifying the amino acid sequence of wild-type BsNTR-WT; the amino acid sequence of wild-type BsNTR-WT is shown in SEQ ID NO. 1; the mutation sites of the nitroreductase BsNTR mutant include A2E, D3E, S30E, L46F, N96D, R102K, Q109K, M110L, H112D, Y115L, G117K, S120K, K121E, T122I, E124N, E136D, K145R, Q160M, Q172D, H175K, R178K, V203T, R204K, D205E, R207A, T210V, S212P, and A213P.

[0007] The present invention provides a polynucleotide that specifically encodes the nitroreductase BsNTR mutant as described above.

[0008] As a further optimization of the above invention, the polynucleotide sequence is shown in SEQ ID NO.3.

[0009] The present invention provides a recombinant plasmid, wherein the recombinant plasmid is an expression vector containing any of the polynucleotides described above and capable of correspondingly translating and expressing the nitroreductase BsNTR mutant described above; the expression vector is pET-28a.

[0010] This invention provides an expression system for BsNTR mutants, which is an Escherichia coli BL21(DE3) cell containing any of the recombinant plasmids described above or whose genome integrates the polynucleotides described above.

[0011] This invention provides a method for preparing a BsNTR mutant of nitroreductase, comprising the following steps: (1) Gene sequence was synthesized based on the amino acid sequence of the BsNTR mutant BsNTR-M as shown in SEQ ID NO.2, and then the gene sequence was constructed on an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) is expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The expression product is then purified to obtain the BsNTR mutant BsNTR-M.

[0012] This invention provides an application of the BsNTR mutant as described above, any of the polynucleotides as described above, any of the recombinant plasmids as described above, or the expression system as described above in improving the thermostability or enzyme activity of BsNTR.

[0013] This invention provides an application of a nitroreductase BsNTR mutant in the organic synthesis of aromatic hydroxylamines and aromatic amines, the preparation of tumor therapeutic drugs, biological detection, and the degradation of environmental pollutants.

[0014] The key modification sites of the mutant BsNTR-M target stability hotspots in the protein structure. By replacing amino acid residues, hydrogen bonding and hydrophobic interactions are enhanced, thereby achieving a synergistic improvement in thermal stability and activity.

[0015] The beneficial effects of this invention are as follows: By designing and modifying the wild-type nitroreductase BsNTR-WT from Bacillus subtilis, the resulting mutant BsNTR-M not only exhibits significantly improved thermal stability (Tm value increased by 14℃ compared to wild-type BsNTR-WT), but also demonstrates 1.6 times enhanced enzyme catalytic activity. This effectively overcomes the technical bottlenecks of poor thermal stability, weak environmental tolerance, and limited activity of the wild-type enzyme. Furthermore, it possesses excellent expression and purification performance, enabling efficient expression and purification in prokaryotic expression systems. Simultaneously, it retains high specificity for nitro compounds and good biosafety and environmental compatibility, allowing for large-scale production without complex equipment. Attached Figure Description

[0016] Figure 1 Results of low-level expression detection of wild-type BsNTR-WT and its mutant BsNTR-M protein; Figure 2 Results of thermostability determination of wild-type BsNTR-WT and its mutant BsNTR-M protein; Figure 3 Results of activity identification and detection of wild-type BsNTR-WT and its mutant BsNTR-M protein; Figure 4 The results show the affinity purification of wild-type BsNTR-WT and its mutant BsNTR-M proteins. Figure 5 The results of QC testing for wild-type BsNTR-WT and its mutant BsNTR-M protein. Detailed Implementation

[0017] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0018] 1. Materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art. Where specific conditions are not specified, they shall be performed according to conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0019] 2. Method 2.1 Construction of the recombinant nitroreductase BsNTR plasmid This invention provides a wild-type nitroreductase (hereinafter referred to as BsNTR-WT) and its mutant (hereinafter referred to as BsNTR-M). BsNTR-WT is derived from the genus Bacillus subtilis, Uniprot number: O34475. The gene sequences of both BsNTR-WT and BsNTR-M were obtained through gene synthesis. The amino acid sequences of BsNTR-WT and BsNTR-M are shown in SEQ ID NO. 1-2, respectively, and the gene sequence of the mutant BsNTR-M is shown in SEQ ID NO. 3. The mutation sites of the mutant BsNTR-M include A2E, D3E, S30E, L46F, N96D, R102K, Q109K, M110L, H112D, Y115L, G117K, S120K, K121E, T122I, E124N, E136D, K145R, Q160M, Q172D, H175K, R178K, V203T, R204K, D205E, R207A, T210V, S212P, and A213P.

[0020] The synthesized genes of BsNTR-WT and BsNTR-M were constructed into the expression vector pET-28a. To facilitate protein purification, a Strep II-8His-TEV-GG tag was added to the N-terminus of the sequence shown in SEQ ID NO. 1-2. The tag sequence is shown in SEQ ID NO. 4 (where StrepII and 8His are tag sequences used for affinity purification, "TEV" is the TEV protease cleavage site used for tag removal during subsequent purification, and "GG" is the tag sequence). The constructed recombinant nitroreductase plasmids were sequenced and verified to be completely identical to the target sequences.

[0021] 2.2 Low-level expression test of nitroreductase BsNTR 2.2.1 Low-level expression of nitroreductase BsNTR The low-level expression of nitroreductase BsNTR was performed using a cell-free expression method. The cell-free expression protocol is described in [Levine, MZ, et al. (2019). Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology]. It mainly includes the following two steps: (1) Preparation of crude extract of Escherichia coli: BL21(DE3) bacterial culture was inoculated into 2×YT medium and cultured at 37℃ until OD 600 When the OD600 reaches 0.6-0.8, add isopropyl β-D-1-thiogalactopyranoside (IPTG). When OD600≈3, centrifuge to collect the bacteria. Wash each gram of phage three times with S30 buffer (10 mM Tris-acetate, pH 8.2, 14 mM magnesium acetate, 50 mM potassium acetate, 2 mM DTT) at 4°C. Resuspend the cells in 1 mL of S30 buffer at a ratio of 1 g of cells. After sonicating the cells with an ultrasonic disruptor, centrifuge at 13000×g for 10 minutes. Transfer the supernatant to a nuclease-free tube, flash freeze in liquid nitrogen, and store at -80°C.

[0022] (2) Expression of protein in cell-free system: Plasmid, cell extract, reaction buffer (phosphoenolpyruvate, PEG mixture, potassium glutamate, magnesium glutamate, amino glutamate, 20 amino acids, reaction energy substances (nicotinamide adenine dinucleotide, adenosine 5'-triphosphate disodium salt, cytidine disodium salt, guanosine 5'-monophosphate disodium hydrate and uridine disodium salt, leucovorin calcium salt and tRNA), glucose, spermidine and 1,4-diaminobutane, etc., were added to a 15 mL nuclease-free centrifuge tube according to the proportions in the above literature. The reaction system was 200 μL. The reaction conditions were 37℃, 200 rpm. After 4 hours, the reaction solution was centrifuged at 12000 rpm for 10 minutes. The supernatant and precipitate were collected and the samples from each step were fixed with loading.

[0023] 2.2.2 Small-scale purification of nitroreductase BsNTR After expression in the cell-free system, 50 μL of Strep-Tactin XT packing material, which had been treated with buffer A (25 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM TCEP), was added to the supernatant. The mixture was incubated at 4°C for 30 minutes. The incubated samples were centrifuged at 12000 rpm at 4°C for 10 minutes. 1 mL of buffer was added, and the samples were washed 3 times. Then, 100 μL of buffer A containing 75 mM biotin was added and incubated with the packing material for 10 minutes. The mixture was centrifuged at 12000 rpm at 4°C for 5 minutes. The eluted samples were collected. A small amount of the samples from each step was fixed with loading buffer and then analyzed by SDS-PAGE.

[0024] The experimental results are shown in Figure 1 Both BsNTR-WT and BsNTR-M proteins were clearly expressed. Small amounts of purified eluted samples were collected for subsequent thermostability and activity tests.

[0025] 2.3 Thermostability test of nitroreductase BsNTR The thermal stability assay for nitroreductase BsNTR uses protein thermal shift (ThermoFluor) technology. Utilizing the structural characteristics of proteins, proteins possess hydrophobic regions hidden internally. As temperature rises, this structure opens up, exposing the hydrophobic regions. The fluorescent dye SYPRO Orange binds to these regions, stimulating fluorescence. Changes in fluorescence intensity form a melting curve; the temperature corresponding to the maximum derivative of the melting curve is the melting point (Tm). The more stable the protein, the higher the measured Tm value.

[0026] The specific procedure for determining the thermostability of nitroreductase BsNTR is as follows: Add 10 µL of nitroreductase BsNTR to each well of a 96-well PCR plate, and then add 10×SYPRO Orange fluorescent dye (Thermoscientific, S6650) to the corresponding wells. Place the 96-well PCR plate in a qPCR instrument, set the instrument parameters, and increase the temperature from 20 °C to 99 °C at a gradient of 1 °C per minute. Calculate the protein melting curve.

[0027] The results are as follows Figure 2 As shown, the Tm value of BsNTR-M is 67.3℃, and the Tm value of BsNTR-WT is 53.3℃. Compared with BsNTR-WT, the thermal stability value of BsNTR-M increased by 14℃, indicating that BsNTR-M has higher stability than BsNTR-WT.

[0028] 2.4 Activity test of nitroreductase BsNTR (1) Prepare buffer: 100 mM sodium phosphate, pH 7.0. The substrate is 0.5 mM nitrofurazone (NFZ) and 1.5 mM NADH. Sample preparation: 1000 nM nitroreductase BsNTR.

[0029] (2) The enzyme activities of BsNTR-WT and its mutant BsNTR-M were determined. 20 μL of Nitrofurazone (NFZ) was transferred to a 384-well plate with two replicates. Then, 20 μL of NADH was added to the corresponding wells of the 384-well plate, and the plate was pre-incubated at 25°C for 5 minutes. After incubation, 20 μL of nitroreductase BsNTR sample was added, thoroughly mixed, and immediately centrifuged and vortexed. The reaction was carried out at 25°C. The absorbance was measured using an M5 microplate reader at 420 nm, and the absorbance changes were recorded. Data analysis was performed using GraphPad Prism9 software to obtain the enzyme activity parameters of the tested protease.

[0030] The results are as follows Figure 3 As shown, the enzyme activity of mutant BsNTR-M was 0.0349 OD420 / min, while that of BsNTR-WT was 0.02247 OD420 / min. The enzyme activity of BsNTR-M was 1.6 times higher than that of WT, indicating that the enzyme activity of BsNTR-M was significantly improved.

[0031] 2.5 Large-scale expression and purification of nitroreductase BsNTR 2.5.1 Expression of nitroreductase BsNTR After transforming the BsNTR-WT and its mutant BsNTR-M recombinant plasmids constructed in step 2.1 into BL21(DE3) strain, the strain was inoculated into 50 ml LB liquid medium and cultured overnight at 37°C. The overnight cultured bacteria were then inoculated into 1 L LB liquid medium at a ratio of 1:100 and cultured at 37°C until the OD600 of the bacterial culture reached 0.6-0.8. 0.5 mM IPTG was then added and cultured overnight at 15°C. The bacterial cells were collected by centrifugation at 5000 rpm for purification.

[0032] 2.5.2 Purification of nitroreductase BsNTR (1) Affinity chromatography The collected bacterial cells were weighed and added to a lysis buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol) at a 1:10 ratio. The cells were homogenized using a high-pressure homogenizer, and the supernatant was collected by centrifugation at 16,000 rpm. Proteins were enriched and purified using a Ni Bestarose FF affinity chromatography column. The specific procedure was as follows: the Ni Bestarose FF affinity chromatography column was first equilibrated with lysis buffer to 10 column volumes. Then, the lysis supernatant was loaded onto the Ni Bestarose FF affinity chromatography column, and gradient elution was performed with lysis buffer containing imidazole. The eluted protein was collected and analyzed by SDS-PAGE. The purification results are shown in the figure below. Figure 4 The eluted sample after affinity chromatography was then added with a certain amount of TEV enzyme protein and digested overnight at 4°C. The protein was then further purified by enrichment using a Ni Bestarose FF affinity chromatography column.

[0033] (2) Gel filtration chromatography and QC detection To obtain proteins with higher purity, the eluted samples were concentrated to approximately 2 mL and then subjected to gel filtration chromatography. The gel chromatography column was a HiLoad 16 / 600 Superdex 75 pg, and the buffer consisted of 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% glycerol. The gel filtration samples were collected and subjected to protein content analysis, specifically SDS-PAGE purity determination, mass spectrometry analysis, and analytical molecular sieve detection.

[0034] Test results as follows Figure 5 As shown in the SDS-PAGE results, the purity of the nitroreductase mutant BsNTR-M protein is high, exceeding 99%.

[0035] Mass spectrometry results also showed that the molecular weight of the sample was basically consistent with that of the target protein, indicating that the purified protein was the target protein. In addition, the results of analytical molecular sieve analysis showed that BsNTR-WT and its mutant BsNTR-M proteins had good aggregation and uniformity, and were in a dimer state in solution.

[0036] 3. Conclusion The above description shows that the present invention provides a recombinant nitroreductase mutant BsNTR-M, which is designed and modified based on the sequence of BsNTR-WT derived from Bacillus subtilis. Compared with BsNTR-WT, it has higher enzyme activity and better protein stability, and therefore has a wider range of application conditions and stronger practical application value, making it more suitable for large-scale production and industrial use.

[0037] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A BsNTR mutant of nitroreductase, characterized in that, The nitroreductase BsNTR mutant is BsNTR-M; the amino acid sequence of BsNTR-M is shown in SEQ ID NO.

2.

2. The BsNTR mutant of nitroreductase according to claim 1, characterized in that, The nitroreductase BsNTR mutant was obtained by modifying the amino acid sequence of wild-type BsNTR-WT; the amino acid sequence of wild-type BsNTR-WT is shown in SEQ ID NO.

1.

3. A polynucleotide, characterized in that, The polynucleotide encodes the BsNTR mutant of nitroreductase as described in any one of claims 1-2; the polynucleotide sequence is shown in SEQ ID NO.

3.

4. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotide as described in claim 3 and capable of expressing the nitroreductase BsNTR mutant as described in claim 1.

5. The recombinant plasmid according to claim 4, characterized in that, The expression vector is pET-28a.

6. An expression system for a BsNTR mutant, characterized in that, The cells are Escherichia coli BL21(DE3) cells containing the recombinant plasmids as described in claims 4-5 or whose genomes integrate the polynucleotides as described in claim 3.

7. A method for obtaining the BsNTR mutant of nitroreductase as described in claim 1, characterized in that, Includes the following steps: (1) Gene sequence was synthesized based on the amino acid sequence of the BsNTR mutant BsNTR-M as shown in SEQ ID NO.2, and then the gene sequence was constructed on an expression vector to obtain a recombinant plasmid; (2) The recombinant plasmid obtained in step (1) was expressed using the Escherichia coli prokaryotic expression system to obtain the expression product. The expression product was purified to obtain the BsNTR mutant BsNTR-M.

8. The use of the BsNTR mutant of nitroreductase as described in claim 1, the polynucleotide as described in claim 3, the recombinant plasmid as described in any one of claims 4-5, or the expression system as described in claim 6 in improving the thermostability or enzyme activity of BsNTR.

9. The application of the BsNTR mutant of nitroreductase as described in claim 1 in the organic synthesis of aromatic hydroxylamines and aromatic amines, the preparation of tumor therapeutic drugs, biological detection and degradation of environmental pollutants.

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